The response of endocrine regulated cells depends on both the binding and the degradation of regulatory hormones. Although the degradation of insulin has been extensively studied with whole animals, perfused organs, suspension of cells and purified enzymes, it is still not clear how insulin is degraded. The major enzyme that splits insulin into its A and B chains is glutathione-insulin transhydrogenase (GIT). To investigate the role of this enzyme in insulin degradation, the principal investigator has purified GIT from mouse liver and lymphoid tissue, produced specific antibodies to it, and set up a specific radioimmunoassay. In the proposed research the antibodies to GIT will be used to investigate the role of this enzyme in insulin degradation. First, the ability of the antibodies to inhibit GIT activity will be studied with isolated fat cells, pancreatic acini and cultured liver cells. These cells will be incubated with 125I-insulin in both the absence and presence of anti-GIT antibodies and insulin degradation will be measured. Second, the antibodies will be used to absorb the supernatants of cells to determine if GIT is secreted and plays a role in insulin degradation. Third, the radioimmunoassay will be used to quantify the cellular levels of GIT and the subcellular location of this enzyme. Finally, the possibility that insulin degradation is related to the action of insulin will be tested by determining the ability of anti-GIT antibodies to inhibit the action of insulin.